Brucei-group trypanosomes are medically important parasites and useful for study of the roles of the mitochondrial genetic system. These cells can suppress development of the mitochondrial respiratory system and this ability can be manipulated by the investigator. Dyskinetoplastic mutant brucei-group trypanosomes have grossly altered mitochondrial DNA, can grow in the vertebrate host but cannot develop a mitochondrial respiratory system. I propose to determine the genome size of normal and mutant mitochondrial DNA and reveal the mutant DNA alterations in Trypanosoma brucei. Normal and mutant DNA will be analyzed by a variety of biochemical and biophysical techniques including equilibrium and velocity sedimentation, thermal denaturation profiles, and renaturation kinetics using hydroxyapatite. These experiments will measure normal and mutant mitochondrial genome size and reveal alterations in base composition, sequence distribution, or intermolecular association in the mutant mitochondrial DNA as well as sequence loss or the production of repeated sequences. Molecular hybridization studies utilizing normal and mutant mitochondrial DNAs, DNA fractions obtained following partial reassociation, or mitochondrial DNAs including mitochondrial ribosomal and transfer RNAs are proposed to reveal the loss or alteration of specific sequences in the mutant mitochondrial DNA. Such studies are also designed to reveal the localization of specific mitochondrial DNA sequences in specific subfractions of the T. brucei mitochondrial DNA which contains two structurally distinct molecular species. The information derived from these studies will be invaluable in understanding the roles of the mitochondrial genetic system, which is impaired in the dyskinetoplastic mutant and essential to the transmission and survival of the cells. The mitochondrial system of brucei-group trypanosomes provides the best available system to test the generality of findings derived from other mitochondrial systems such as yeast.